HIV-envelope-specific DARPin-based microbicide strategies In order to be a viable candidate microbicide, an anti-HIV strategy must be highly effective, block cell-free and cell-associated infections in relevant in vitro model systems, prevent in vivo infection without perturbing immune function or causing any toxic reactions, and be able to be produced at low costs. Through a recently established collaboration, we identified a novel, highly specific approach to inhibit HIV infection using the Designed Ankyrin Repeat Protein (DARPin) technology. The initial proof of concept studies documented the selection of CD4- specific DARPins that are highly specific and efficiently prevent in vitro infection with a wide range of HIV isolates as well as SIV at nanomolar levels, without interfering with basic cellular functions. DARPins possess unique characteristics exhibiting exceptional stability and specificity, and cost little to produce. Combining these features with the ability to prevent HIV infection, DARPins represent a promising novel strategy to explore for the prevention of HIV spread. Expanding on this new collaborative research, we propose to select highly active HIV Env-specific DARPins that will be able to prevent mucosal HIV infection. We hypothesize that cocktails of Env-specific DARPins, which recognize gp120 and gp41, will (i) efficiently prevent in vitro cell-free and cell-associated infection with numerous HIV and SIV isolates, functioning in the low nanomolar range, (ii) not impair innate or adaptive immune functions, and (iii) prevent mucosal infection with SHIV162P3 in macaques, with no adverse impact on local immune function, flora, and pH. This innovative anti-HIV technology is being developed in Dr. Trkola's laboratory (in collaboration with Dr. Pl[unreadable]ckthun's group) and, once selected through rigorous screening assays, promising candidates will be thoroughly tested in a variety of in vitro viral and immune systems using relevant cell and tissue-based assays established in both Drs. Robbiani's and Trkola's laboratories. The lead novel candidate(s) will then be tested for in vivo activity in macaques through Dr. Robbiani's laboratory (in collaboration with Dr. Blanchard). Utilizing a novel technology to develop an anti-HIV Env specific strategy, this high-risk DARPins approach should yield critical advances towards preventing HIV infection and spread. Project Summary-Relevance: These studies will provide the first data on the efficacy of this novel topical microbicide to prevent vaginal infection in vivo. With proof of concept that this represents a promising strategy to tackle HIV at the mucosa, we will be able to explore application of this approach to coitally independent modalities, as well as for the potential application against rectal transmission. These data will advance the design and future testing of this novel technology to improve microbicide strategies to test in women and men.